US4070495A - Microscope slide - Google Patents

Microscope slide Download PDF

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Publication number
US4070495A
US4070495A US05/672,572 US67257276A US4070495A US 4070495 A US4070495 A US 4070495A US 67257276 A US67257276 A US 67257276A US 4070495 A US4070495 A US 4070495A
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United States
Prior art keywords
methylene blue
colored
dyestuffs
microscope slide
dyestuff
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US05/672,572
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English (en)
Inventor
Dieter Berger
Werner Guthlein
Wolfgang Werner
Peter Rieckmann
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Roche Diagnostics GmbH
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Boehringer Mannheim GmbH
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Publication date
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • This invention relates to a pre-colored microscope slide for blood investigations.
  • the differential blood count is one of the most frequently carried out microscopic methods.
  • the individual blood particles erythrocytes, leukocytes, thrombocytes, etc.
  • dyestuffs which permit a microscopic differentiation and the recognition of pathological changes.
  • the previously employed staining processes involve several steps, for example, preparing a blood smear, fixing, in some cases several stainings, washing and drying and are, therefore, very laborious.
  • the quality of the staining depends very considerably upon the quality of the dyestuffs employed, as well as upon the training and experience of the personnel involved.
  • the stainings are generally dull, diffuse and lacking in contrast and, in addition, the three different forms of the granulated leukocytes (eosinophils, neutrophils and basophils) can only be differentiated with difficulty.
  • FIG. 1a and 1b are top plane and side views, respectively, of a microscopic slide according to the invention.
  • the methylene blue N is preferably used in the form of its monohydrochloride instead of the commercially available zinc double salt.
  • German Pat. No. 2,153,673 gives no indication of the quality of the dyestuffs used.
  • pre-colored microscope slides are obtained, the staining properties of which correspond to those of this German Patent (see pages 5 -6 thereof).
  • Methylene blue N (new methylene blue, C.I. Basic Blue 24, 3,7-bis-(N-ethylamino)-2,8-dimethylphenothiazonium chloride) is commercially available in about 50 - 70% purity and, according to our own findings, thin layer chromatographic analysis shows three to four additional spots in varying amounts. The remainder is zinc chloride and sodium chloride.
  • an excellent purification of methylene blue N can be carried out by dissolving this dyestuff in water and precipitating out the hydrochloride thereof with hydrochloric acid. There is thus obtsined a chormatographically pure product which only contains about 0.5% zinc chloride and is in the form of the monohydrochloride.
  • Cresyl violet acetate (Cresylechtviolett, Cresyl Fast Violet acetate, 5,9-diaminobenzo (a)phenoxazonium acetate) is commercially available, contaminated with more or less large amounts of sodium acetate. In this case, too, thin layer chromatographic analysis shows three additional spots in varying amounts. Purification of this dyestuff can be carried out in the following manner: The sodium acetate is first removed by careful digestion with water. Thereafter, the dyestuff is dissolved in methanol and precipitated out with diethyl ether. Repetition of this procudure gives a chromatographically pure product.
  • the dyestuffs purified in the above-described manner dissolve in methanol without leaving a residue and can be applied to microscope slides in the manner described on page 4 of the above-mentioned German Pat. No. 2,153,673. Furthermore, according to the teachings in German Pat. No. 2,424,955, they can be dissolved in water and applied, together with polyoxyethylene-sorbitan monopalmitate (Tween 80)
  • the dyestuff solutions are sprayed on to microscope slides in the manner described in co-pedning application Ser. No. 672,574 filed Mar. 31, 1976 (German Patent Application No. P 25 15 869.4).
  • the purified dyestuffs are preferably applied to the microscope slides in total amounts of about 0.5 to 10 ⁇ /cm 2 .
  • the ratio of methylene blue N to cresyl violet acetate may thereby vary between the values of 1:1.5 to 1:5.
  • the pre-coated microscope slides according to the present invention can, as described on pages 4 to 5 of German Pat. No. 2,153,673, be provided with a drop of blood and then covered with a cover slip; after about 3 to 5 minutes, microscopic examination can be carried out witha an oil immersion objective.
  • the granula of the eosinophils have a luminescent yellow color, in comparison with the there-described orange coloration.
  • the granula of the basophils are orange-red colored, in comparison with the there-described purple coloration.
  • the orange coloration which is the most effectively identifiable characteristic, there only occurs on the edge of the cells in the case of suitable focussing.
  • a further advantage of the microscope slides according to the present invention is that the stainings no longer depend upon variations of quality of commercially available dyestuffs. In the case of the large-scale production of large numbers of microscope slides, the reproducibility with regard to dyestuff application and composition is of the greatest possible importance.
  • the microscope slides can be made from glass. However, insofar as the dyestuffs are sprayed on in the manner described in co-pending Application Ser, No. 672,574 (German Patent Application No. P 25 15 869.4), synthetic resin microscope slides can also be used, whereby the dyestuff can, of course, also be applied to cover slips made from glass or synthetic resin.
  • cresyl violet acetate obtained from Matheson, Coleman & Bell
  • 50 g. cresyl violet acetate obtained from Matheson, Coleman & Bell
  • the dyestuff was filtered off with suction and washed twice with 100 ml. amounts of ice-cold water.
  • the filter residue was dissolved, with warming, in 1.6 liters methanol and insoluble material removed by suction filtration at about 30° C.
  • the filtrate was slowly mixed, while stirring, with 3.5 liters diethyl ether and then further stirred for 30 minutes, while cooling with ice.
  • the crystals formed were filtered off with suction, dissolved, with warming, in 620 ml. methanol and the solution is cooled to 30° C.
  • the dyestuff was precipitated out by the addition of 1.85 liters diethyl ether, while stirring and cooling with ice. After filtering off with suction and washing three times with 120 ml. amounts of diethyl ether-methanol (3:1), there were obtained, after drying over phosphorus pentoxide, 22 g. cresyl violet acetate in the form of dark green crystals. This product contains, by weight, about 82% violet acetate, 8% cresyl violet chloride and 10% water. Upon heating to 150° C., decomposition occurs. According to chromatographic investigation (DC finished plate, silica gel 60 F 254, Merck; elution agent system n-butanol-glacial acetic acid-water 4:1:5), the substance was practically pure; R F value: 0.6.
  • the purified dyestuff contains, by weight, 95 - 97% methylene blue N chloride hydrochloride, as well as 3 - 5% water. Zinc was only present in traces. According to chromatographic investigation (DC finished plate silica gel 60 F 254, Merck; elution agent system n-butanol-glacial acetic acid-water 4:1:5), the dyestuff was practically pure; R F value: 0.5)
  • a solution was prepared of the following composition, using the purified dyestuffs according to Examples 1 and 2:
  • cresyl violet acetate 270 mg.
  • This solution was coated as a film, using a swab of cottonwool, on to a microscope slide and then dried or was sprayed from a 0.5 mm. wide spray nozzle (SS 60 67228-45 of the firm Spraying Systems) with a spraying angle of about 25° at a distance of 20 cm. through a mask in a breadth of 3 cm. on to a microscope slide which passed below the nozzle at a speed of 1.5 meters/minute.
  • the spray pressure was 1.2 ats. and the rate of flow through the nozzle was 10 ml./minute.
  • the amount of dyestuff applied is about 3 ⁇ g./cm 2 .
  • a drop of blood with a volume of about 5 - 10 ⁇ l. was applied to the pre-colored microscope slide and then covered with a cover slip. After about 3 to 5 minutes, the staining was assessed under a microscope at about 800 fold magnification, using an oil immersion objective.
  • the individual particles of the blood have the following stainings:
  • reticulocytes purple-colored reticulum within the scarcely colored erythrocytes.
  • neutrophils purple-colored nucleus within a fine granulated, almost colorless plasma.
  • eosinophils purple-colored nucleus within a coursely granulated yellow plasma.
  • basophils dark purple-colored nucleus within an average-sized, compact granulated orange-red colored plasma.
  • lymphocytes purple-colored nucleus with bright purple-colored plasma.
  • monocytes like lymphocytes but larger and with more plasma.
  • thrombocytes small purple-colored particles.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Microscoopes, Condenser (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
US05/672,572 1975-04-11 1976-03-31 Microscope slide Expired - Lifetime US4070495A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2515966A DE2515966C3 (de) 1975-04-11 1975-04-11 Vorgefärbte Objektträger für die Blutunte rsuchung
DT2515966 1975-04-11

Publications (1)

Publication Number Publication Date
US4070495A true US4070495A (en) 1978-01-24

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ID=5943661

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US05/672,572 Expired - Lifetime US4070495A (en) 1975-04-11 1976-03-31 Microscope slide

Country Status (23)

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US (1) US4070495A (enrdf_load_stackoverflow)
JP (1) JPS51126893A (enrdf_load_stackoverflow)
AR (1) AR210757A1 (enrdf_load_stackoverflow)
AT (1) AT350190B (enrdf_load_stackoverflow)
BE (1) BE840454A (enrdf_load_stackoverflow)
CA (1) CA1052675A (enrdf_load_stackoverflow)
CH (1) CH621629A5 (enrdf_load_stackoverflow)
CS (1) CS205019B2 (enrdf_load_stackoverflow)
DD (1) DD123628A5 (enrdf_load_stackoverflow)
DE (1) DE2515966C3 (enrdf_load_stackoverflow)
DK (1) DK145318C (enrdf_load_stackoverflow)
ES (1) ES446836A1 (enrdf_load_stackoverflow)
FI (1) FI59677C (enrdf_load_stackoverflow)
FR (1) FR2307271A1 (enrdf_load_stackoverflow)
GB (1) GB1480576A (enrdf_load_stackoverflow)
HU (1) HU176928B (enrdf_load_stackoverflow)
IE (1) IE42552B1 (enrdf_load_stackoverflow)
IT (1) IT1059617B (enrdf_load_stackoverflow)
LU (1) LU74738A1 (enrdf_load_stackoverflow)
NL (1) NL7603628A (enrdf_load_stackoverflow)
PL (1) PL107576B1 (enrdf_load_stackoverflow)
SE (1) SE420355B (enrdf_load_stackoverflow)
SU (1) SU904536A3 (enrdf_load_stackoverflow)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4185085A (en) * 1976-11-09 1980-01-22 Boehringer Mannheim Gmbh Differential diagnostic sperm examination
US4193980A (en) * 1978-01-05 1980-03-18 Corning Glass Works Dry preparation for reticulocyte staining
US4248821A (en) * 1979-07-25 1981-02-03 Dellen Adrian F Van Method and device for embedding a specimen for microscopic examination
US4606950A (en) * 1984-08-31 1986-08-19 Ellen M. Corbet Method for assembling a floral arrangement
US5106744A (en) * 1990-11-01 1992-04-21 Cytocolor Inc. Method of staining monocytes and compositions thereof
US20210181085A1 (en) * 2017-10-26 2021-06-17 Essenlix Corporation Rapid measurement of platelets

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4400370A (en) * 1980-03-12 1983-08-23 Lawrence Kass Metachromatic dye sorption means for differential determination of leukocytes
US4581223A (en) * 1980-03-12 1986-04-08 Lawrence Kass Individual leukocyte determination by means of differential metachromatic dye sorption
CA1155041A (en) * 1980-04-21 1983-10-11 Michael E. Jolley Fluorescent nucleic acid stains
US4500509A (en) * 1981-03-11 1985-02-19 Lawrence Kass Metachromatic dye sorption and fluorescent light emmisive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes
DK1799662T3 (da) 2004-09-23 2013-07-15 Wista Lab Ltd Fremgangsmåder til kemisk syntese og oprensning af diaminophenothiaziniumforbindelser, herunder methylthioniniumchlorid (MTC)
MY162313A (en) 2006-07-11 2017-05-31 Wista Lab Ltd Methods of synthesis and/or purification of diaminophenothiazinium compounds

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3906120A (en) * 1970-10-30 1975-09-16 Gen Electric Method for preparing slides for blood evaluation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT966513B (it) * 1970-10-30 1974-02-20 Gen Electric Vetrini precolorati per esami del sangue
US3796594A (en) * 1970-10-30 1974-03-12 Gen Electric Stain coated slides for differentially staining blood
GB1473945A (en) * 1973-05-24 1977-05-18 Gen Electric Method for preparing slides for blood evaluation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3906120A (en) * 1970-10-30 1975-09-16 Gen Electric Method for preparing slides for blood evaluation

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4185085A (en) * 1976-11-09 1980-01-22 Boehringer Mannheim Gmbh Differential diagnostic sperm examination
US4193980A (en) * 1978-01-05 1980-03-18 Corning Glass Works Dry preparation for reticulocyte staining
US4248821A (en) * 1979-07-25 1981-02-03 Dellen Adrian F Van Method and device for embedding a specimen for microscopic examination
US4606950A (en) * 1984-08-31 1986-08-19 Ellen M. Corbet Method for assembling a floral arrangement
US5106744A (en) * 1990-11-01 1992-04-21 Cytocolor Inc. Method of staining monocytes and compositions thereof
US20210181085A1 (en) * 2017-10-26 2021-06-17 Essenlix Corporation Rapid measurement of platelets

Also Published As

Publication number Publication date
AT350190B (de) 1979-05-10
CA1052675A (en) 1979-04-17
AU1267776A (en) 1977-10-13
DE2515966C3 (de) 1979-11-22
FR2307271B1 (enrdf_load_stackoverflow) 1980-03-28
AR210757A1 (es) 1977-09-15
JPS51126893A (en) 1976-11-05
IT1059617B (it) 1982-06-21
DE2515966A1 (de) 1976-10-21
DE2515966B2 (de) 1979-03-22
IE42552L (en) 1976-10-11
FR2307271A1 (fr) 1976-11-05
FI59677B (fi) 1981-05-29
FI59677C (fi) 1981-09-10
LU74738A1 (enrdf_load_stackoverflow) 1976-11-11
ATA262076A (de) 1978-10-15
DK149876A (da) 1976-10-12
PL107576B1 (pl) 1980-02-29
DD123628A5 (enrdf_load_stackoverflow) 1977-01-05
IE42552B1 (en) 1980-08-27
DK145318C (da) 1983-04-05
DK145318B (da) 1982-10-25
FI760911A7 (enrdf_load_stackoverflow) 1976-10-12
CS205019B2 (en) 1981-04-30
GB1480576A (en) 1977-07-20
NL7603628A (nl) 1976-10-13
HU176928B (hu) 1981-06-28
SU904536A3 (ru) 1982-02-07
ES446836A1 (es) 1977-06-01
SE7602728L (sv) 1976-10-12
SE420355B (sv) 1981-09-28
CH621629A5 (enrdf_load_stackoverflow) 1981-02-13
BE840454A (fr) 1976-10-07

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